Method for treating a subject afflicted with intestinal malabsorption

ABSTRACT

A composition for human or animal consumption for supplying folate which includes a natural isomer of reduced folate, such as (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolic acid, and their polyglutamyl derivatives is disclosed. Such compositions include multivitamin preparations (with or without minerals and other nutrients); breakfast foods such as prepared cereals, toaster pastries and breakfast bars; infant formulas; dietary supplements and complete diet and weight-loss formulas and bars; animal feed (for example pet foods) and animal feed supplements (such as for poultry feed). The amount of the natural isomer of a reduced folate in a composition for human consumption can range between about 5% and about 200% of the daily requirement for folic acid per serving or dose.

[0001] This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/010,898, filed Jan. 31, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field ofnutrition, and more specifically to food and vitamin preparationscontaining the natural isomer of reduced folates.

BACKGROUND OF THE INVENTION

[0003] The folates are ubiquitous to nearly all forms of life. Humansand many other animals lack the capacity to make their own folate whichthus is an essential vitamin, one type of essential nurient. Anemiaespecially during pregnancy and in the geriatric population was an earlyindication of a dietary requirement for folate. A major function offolate is to remove one-carbon units from molecules being metabolizedand then deliver them to molecules being synthesized. As an example,folate participates in the formation of the nucleic acids. Further, theactivity of DNA is controlled, in part, by methylation, and the primarymethylating agent of the body (S-adenosylmethionine) is made in ametabolic cycle involving a folate. Many studies have, therefore,focused on the relationship of folate status to cancer susceptibility,especially colorectal adenoma.

[0004] The importance of folate to proper growth is clearly evident inthe occurrence of neural tube defects in newborn infants. Reports fromseveral countries have shown that a majority of such cases areassociated with low folate levels in the mother. The incidence of thesedefects as well as of cleft lip/palate is considerably reduced whenwomen are given folic acid (I) starting early in pregnancy. Recently, asignificant correlation has been discovered between vitamin deficiency,especially of folate, and peripheral vascular disease, a major cause ofdeath. A high percentage of individuals with this affliction haveabnormal blood levels of homocysteine, a precursor to methionine in thefolate dependent step of the S-adenosylmethonine cycle. Folatedeficiency has also been linked to defective maturation of a number ofdifferent cell types, to nervous system disorders, and to decreasedimmune response.

[0005] The clear relation of folate intake to health has caused manygovernmental agencies around the world (such as the U.S. NationalResearch Council) to specify a recommended dietary allowance (“RDA”) forfolate. In the U.S. these values are used by the Food and DrugAdministration to establish the Reference Daily Intake (“RDI”) that islisted on food labels, currently 0.4 mg for adults. The highest dailyamount of folic acid recommended by a country is 2.0 mg for healthyadults. Many products are available that contain RDI or near RDI levelsof folic acid (I) including most daily multiple vitamins. These can bepurchased in solid (e.g. tablet, capsule, or powder) or in liquidformulations, both over-the-counter and by prescription. In the U.S.folic acid (I) is also available by itself typically at a dosage of 0.4mg, but also up to 0.8 mg in health food stores. Many complete diets,infant diets, dietary supplements and weight loss products also containfolic acid (I). In some countries folic acid is added to specific foodtypes as determined by health officials to provide adequate folate tothe general population without risking excess consumption. Manybreakfast foods, such as cereals, cereal bars, breakfast drink mixes,breakfast bars and toaster pastries have folic acid (I) added at amodest fraction of the RDI, typically 10-50% of the adult value perserving. In many of these uses the folic acid (I) is accompanied byother vitamins, sometimes at RDI dosages, but also at lower or muchhigher levels. Frequently, though not always, essential mineralnutrients are also present. Further, many products also includecompounds hypothesized to have health related value, but which eitherhave not been officially recognized as effective, or for which optimalamounts have not been set. Products such as those described above aremeant to fill an important and wide spread need for folate, especiallyamong those whose dietary habits would otherwise preclude intake of asufficient amount of this vitamin.

[0006] Folic acid (I) is a component of many animal and pet foods. It isalso included in powders or liquids used as animal feed supplements,often in combination with other nutrients. For example, the NationalResearch Council (NRC) recommends diets containing 0.2 mg and 1.0 mg offolic acid (I) per kg of dry diet (assuming 5 kcal metabolizable energyper gram) for dogs and cats, respectively. For chicks the NRC hasrecommended 0.55 mg folic acid per kg of diet, although recentliterature suggests that the optimal value is about three times higherthan this.

[0007] The form of folate currently added to all commercial vitaminpreparations or which is added to foods, folic acid (I) (also known aspteroyl-L-glutamic acid), is not one of the major forms found in naturalfresh foods. The structure of folic acid (I) differs from the mostabundant natural folate in several aspects. First, the side-chain ofnatural folates in almost all fresh foods contains more than oneL-glutamic acid moiety. Frequently, five to seven (but covering aconsiderable span of more or fewer) of this amino acid are linkedtogether into a polyglutamate chain. It is well known, however, that theprimary form by which folates are absorbed has only a single glutamateresidue. Cleavage of the extra glutamates of dietary folates is usuallyaccomplished by an enzyme in the digestive tract. In this aspect folicacid (I) is not at a disadvantage in comparison to naturally occurringfolates.

[0008] The second difference between folic acid (I) and natural folatesis that whereas the pteridine ring of the former (I) is fully oxidized,natural folates in fresh uncooked foods are mostly present as thetetrahydro forms. Almost all of the known physiological functions offolate are performed by tetrahydrofolic acid, (6S)-FH₄ (II), or by a onecarbon derivative of it illustrated as follows: 5-methyl-(6S)-FH₄ (III),5-formyl-(6S)-FH₄ (IV), 10-formyl-(6R)-FH₄ (V), 5,10-methylene-(6R)-FH₄(VI), 5,10-methenyl-(6R)-FH₄ (VII), and 5-formimino-(6S)-FH₄ (VIII). Thestructural formula for each of these compounds is provided below.

[0009] There is no known direct cofactor function for folic acid (I)itself in humans. Some (6S)-tetrahydrofolic acid polyglutamate is foundin plants or animals, but the majority of folate is polyglutamate formsof either 5-methyl-, 5-formyl-(6S)-tetrahydrofolic acid, and in somecases 10-formyl-tetrahydrofolic acid. Presumably, most of the folic acid(I) found in biological food sources results from oxidation, especiallyon storage. When folic acid (I) is absorbed by the digestive tract it iseventually reduced to active (6S)-tetrahydrofolic acid (II) by theenzyme dihydrofolate reductase.

[0010] The oral bioavailability of folic acid (I) has been shown to bewidely variable. The literature contains reports of individuals havingpoor intestinal uptake of folic acid (I) who respond normally tointramuscular injection of folic acid (I), or had normal serum folatestatus prior to any folic acid challenge. Several small scaleinvestigations in which the values have been averaged have concludedthat the oral uptake of several of the reduced folates is similar tofolic acid (I). However, there is reason to believe that a segment ofthe population possesses adequate oral response to reduced folates, butnot to oral folic acid (I).

[0011] 5-Formyl-tetrahydrofolic acid (also known as leucovorin orfolinic acid) has long been used in therapeutic doses for severaldiseases. Examples include rescue from the toxicity of methotrexatechemotherapy, and the synergistic combination with fluorouracil fortreatment of various cancers. It is also given to treat acute anemia notdue to B₁₂ deficiency. 5-Methyl-tetrahydrofolic acid in high doses (forexample, 50 mg/day) has been patented for treatment of depression (andother neurological disorders) (EP382019 and EP388827 to Le Grazie 1990,and EP482493 to Le Greca 1992).

[0012] That reduced folates have been overlooked as an improved sourcefor providing the RDA level is in part due to the stereochemistry ofthese compounds. In addition to the single chiral center of theL-glutamate chain in folic acid (I, the tetrahydrofolates contain asecond stereochemical center at carbon-6. Chemical reduction of folicacid (I) produces a nearly racemic mixture of the two isomers at thisposition. This is in contrast to the reduced folates found in naturewhich all consist of a single diastereoisomer, all having the sameL-configuration at carbon-6. (Compounds II-VIII are shown as the naturalisomer). For many years only the racemic 6(R,S) mixture of5-formyl-tetrahydrofolic acid (leucovorin) has been used for therapy ofdiseases. Recently, however, concern over the possible effects of theunnatural isomer component has resulted in the commercial introductionof the pure natural isomer for these high dose disease treatments byLederie, although at very high cost. Most therapeutic regimes utilizingleucovorin last a few weeks or perhaps months. The effect of a long termexposure to the unnatural isomer of reduced folates is unknown. Forexample, although little 5-formyl-(6R)-tetrahydrofolic acid is absorbed,there is considerable uptake of the unnatural isomer of5-methyl-tetrahydrofolic acid by the intestinal tract and other cells ofthe body which with continuous intake may lead to adverse consequences.

[0013] Until recently, processes for making the natural isomer ofreduced folates have been limited in scale, or costly, or both. Theseinclude chromatographic separation, enzymatic reduction, and fractionalcrystallization. The use of reduced folates as a daily source of vitaminrequires a method that is applicable to large scale production of thenatural isomer having high purity at a cost that will not place a burdenon the average consumer.

SUMMARY OF THE INVENTION

[0014] The present invention relates to a composition which includes oneor more natural isomers of reduced folate and a nutritional substance.The one or more natural isomers of reduced folate is selected from thegroup consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. The nutritional substance is a food preparation, an essentialnutrient preparation, or a combination thereof. When the nutritionalsubstance is a food preparation, the food preparation includes two ormore food components. Each gram of the food preparation has a naturalmolar amount, N, of the one or more natural isomers of reduced folate, Nbeing greater than or equal to zero, and each gram of the compositionhas a total molar amount, T, of the one or more natural isomers ofreduced folate greater than N. When the nutritional substance is anessential nutrient preparation, the essential nutrient preparationincludes a vitamin other than ascorbic acid.

[0015] The present invention also relates to method for increasing thefolate content of a nutritional substance. The method includes providinga nutritional substance selected from the group consisting of a foodpreparation, an essential nutrient preparation, and combinationsthereof. The method further includes incorporating into the nutritionalsubstance a molar amount of one or more natural isomers of reducedfolate selected from the group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. When the nutritional substance is a food preparation, the foodpreparation comprises two or more food components. When the nutritionalsubstance is an essential nutrient preparation, the essential nutrientpreparation comprises a vitamin other than ascorbic acid.

[0016] A significant number of people are folate deficient; especiallyvulnerable are those whose life style does not include sufficient freshfood sources of folates. An object of this invention is nutritionalcompositions in which the natural isomer of tetrahydrofolic acid, or aderivative thereof, is substituted for the usual folic acid (I) for thesatisfaction or partial satisfaction of the dietary requirement for thisvitamin. While some may not be greatly affected by the inclusion ofreduced folates in multivitamin preparations and breakfast foods, stilla substantial number of people, and thus the average health of thepopulation, will be improved by addressing the needs of those for whomfolic acid (I) bioavailability is poor. Consumer confidence with regardto consumption of a food or other nutritional product will be increasedwith the knowledge that the folate content is chemically identical tothe most abundant natural forms of this vitamin, except for theadvantageous absence of multiple glutamate residues. A further advantageis that health agencies will be aided in recommending optimal levelswhen a more uniformly absorbed form of folate is widely used.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to a composition which includes oneor more natural isomers of reduced folate and a nutritional substance.Natural isomers of reduced folate suitable for use in the presentinvention include, for example, (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid, and5-formimino-(6S)-tetrahydrofolic acid. Other natural isomers of reducedfolate suitable for use in the present invention include thepolyglutamyl, such as the diglutamyl, triglutamyl, tetraglutamyl,pentaglutamyl, and hexaglutamyl, derivatives of (6S)-tetrahydrofolicacid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolicacid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, and5-formimino-(6S)-tetrahydrofolic acid. Any or all of the natural isomersof reduced folate can be present in its chirally pure form, or,alternatively, the composition can optionally contain a molar amount ofone or more unnatural isomers of reduced folate, such as(6R)-tetrahydrofolic acid, 5-methyl-(6R)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6S)-tetrahydrofolic acid,5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. The molar amount of the natural isomer of reduced folate can beequal to the molar amount of its corresponding unnatural isomer (aswhere the unnatural and natural isomer are present as a racemicmixture), or, preferably, the natural isomer of reduced folate can bepresent in a molar amount greater than the molar amount of thecorresponding unnatural isomer. The total molar amount of the one ormore natural isomers of reduced folate present in the composition can bebetween 5% and 200% of a human daily requirement for folate per acustomarily consumed quantity of the composition. As used herein, thetotal molar amount of the one or more natural isomers of reduced folateincludes natural isomers of reduced folates which are naturally presentin the nutritional substance as well as natural isomers of reducedfolates which might have been added to the nutritional substance. Thecustomarily consumed quantity of various compositions depends, ofcourse, on the nature of the composition. Where the composition includesa food preparation, the customarily consumed quantity is the amount ofthe food preparation customarily consumed per eating occasion, forexample, as set forth by the U.S. Food and Drug Administration for thepurpose of establishing realistic and consistent serving sizes for usein food labeling. Examples of customarily consumed quantities forvarious food groups can be found in 21 C.F.R. §101.12, which is herebyincorporated by reference.

[0018] The human daily requirement for folate varies from person toperson, depending on factors such as body weight, age, health, sex, andthe like. Suitable values for the human daily requirement for folateinclude RDI and RDA values, promulgated, respectively, by the FDA andthe National Research Council (“NRC”). Presently, RDI values, expressendin terms of micrograms of folic acid (nanomoles of folic acid), are 400μg (907 nmoles) for adults and 800 μg (1814 nmoles) for pregnant women.In view of this, the compositions of the present invention can havebetween 45 and 1814 nnmoles or between 91 and 3625 nmoles of folate percustomarily consumed quantity of the nutritional substance. Current RDAvalues are published in National Research Council: Recommended DailyAllowances, 10th ed., Washington, D.C. (1989), which is herebyincorporated by reference. They are, again expressend in terms ofmicrograms of folic acid (nanomoles of folic acid), 25 μg (57 nmoles)for infants 0-6 months of age, 35 μg (79 nmoles) for infants 6 months toone year of age, 50 μg (113 nmoles) for infants 1-3 years of age, 100 μg(227 nmoles) for infants 7-10 years of age, 200 μg (454 nmoles) for maleadults, 180 μg (408 nmoles) for female adults other than pregnant adultfemales, and 400 μg (907 nmoles) for pregnant females. In view of this,typical compositions of the present invention can have between 2.8 and113 nmoles, between 4 and 159 nmoles, between 28 and 227 nmoles, between11 and 454 nmoles, between 22 and 907 nmoles, between 20 and 816 nmoles,or between 45 and 1812 nmoles of folate per customarily consumedquantity of the nutritional substance. Suitable values for the humandaily requirement for folates are also established by the World HealthOrganization as 7.03 nmoles/kg of body weight. For pregnant women thevalue calculated based upon body weight should be increased by about 454to about 680 nmoles.

[0019] The total molar amount of the one or more natural isomers ofreduced folate present in the composition can, alternatively, be between5% and 3000% of an animal daily requirement for folate per a customarilyconsumed quantity of the nutritional substance. The animal, whose dailyrequirement for folate is referred to above, can be, for example, a dog,a cat, a chicken, a cattle, a domestic animal, a goat, a horse, a mink,a fox, a sheep, or a swine. Suitable values for the an animal's dailyrequirement for folate are promulgated, for example, by the NRC inNutrient Requirements of Domestic Animals (Washington:National AcademyPress), particularly in those publications having the followingsubtitles: “Nutrient Requirements of Beef Cattle,” Seventh RevisedEdition (1996, ISBN 0-309-05426-5); “Nutrient Requirements of Cats,”Revised Edition (1986, ISBN 0-309-03682-8); “Nutrient Requirements ofDairy Cattle,” Sixth Revised Edition, Update (1989, ISBN 0-309-03826-X);“Nutrient Requirements of Dogs, Revised (1985, ISBN 0-309-03496-5);“Nutrient Requirements of Fish,” (ISBN 0-309-04891-5); “NutrientRequirements of Goats: Angora, Dairy, and Meat Goats in Temperate andTropical Countries,” (ISBN 0-309-03185-0); “Nutrient Requirements ofHorses,” Fifth Revised Edition, (1989, ISBN 0-309-03989-4); “NutrientRequirements of Laboratory Animals,” Fourth Revised Edition (1995, ISBN0-309-05126-6); “Nutrient Requirements of Mink and Foxes,” SecondRevised Edition, (1982, ISBN 0-309-03325-X); “Nutrient Requirements ofPoultry,” Ninth Revised Edition, (1994, ISBN 0-309-04892-3); “NutrientRequirements of Sheep,” Sixth Revised Edition, (1985, ISBN0-309-03596-1); and “Nutrient Requirements of Swine,” Ninth RevisedEdition, (1988, ISBN 0-309-03779-4) (collectively referred to as “NRCAnimal Nutrient Requirements”), which are hereby incorporated byreference.

[0020] As indicated above the nutritional substance can be a foodpreparation or an essential nutrient preparation. Essential nutrientpreparations are materials which contain one or more essentialnutrients. Where only one essential nutrient is present in the essentialnutrient preparation, that essential nutrient can be a vitamin otherthan ascorbic acid. The essential nutrient preparation can,alternatively, include a vitamin other than ascorbic acid and, inaddition, ascorbic acid. As used herein, essential nutrients are thosenutients which are required to sustain health but which cannot beeffectively produced by one or more animals or by humans. Examples ofessential nutrients are compiled in a number of published sources,including Modern Nutrition in Health and Disease, 8th ed., Shils et al.,eds., Philadelphia:Lea and Febiger (1994), which is hereby incorporatedby reference. Essential nutients are meant to include essential vitaminsand provitamins thereof, essential fats, essential minerals, such asthose minerals for which daily values have been recommended, andessential amino acids. One example of an essential nutrient preparationis a formulation which contains a vitamin and a caloric content of lessthan 2.5 cal per dry gram, preferably less than 2 cal per dry gram, mostpreferably less than 1.8 cal per dry gram. Essential nutrientpreparations also include those materials which contain at least onevitamin in an amount greater than 15%, preferably greater than 20%, morepreferably greater than 40% of the U.S. adult RDA for that essentialnutrient per gram of essential nutrient preparation. Still othersuitable essential nutrient preparations contain at least two vitamins,each in an amount greater than 10%, preferably greater than 15%, morepreferably greater than 20% of the U.S. adult RDA for that essentialnutrient per gram of essential nutrient preparation. Suitable essentialnutrient preparations are commonly referred to as dietary supplements,vitamin supplements, and mineral supplements, multiple vitaminsupplements, and the like, and are typically available commercially inthe form of pills, tablets, capsules, powders, syrups, and suspensions.Preferably, the essential nutrient composition contains at least oneessential nutrient in an amount greater than 25%, more preferablygreater than 50%, and most preferably greater than or equal to 100% ofthe daily requirement for that essential nutrient per customarilyconsumed quantity of the essential nutrient preparation.

[0021] As indicated above, the nutritional substance can also be a foodpreparation. Food preparations are materials which contain one or moreamino acid, carbohydrate, or fat, which are suitable for human or animalconsumption, and which are not essential nutrient preparations. It ispreferred that the food preparation be a two or more component foodpreparation. For example, a two or more component food preparation canbe a mixture of two or more one-component foods. One component foods arefoods which are derived substantially from a single natural source. Asmall percentage of the one-component food can be derived from a secondnatural source, but that percentage, by weight, is preferably less than5%, more preferably less than than 1%, more preferably less than 0.1%.One component foods include, for example, sugar, unsweetened juice,nectar, or puree from a single species of plant, such as unsweetenedapple juice (including a blend of different varieties of apple juice),grapefruit juice, orange juice, apple sauce, apricot nectar, tomatojuice, tomato sauce, tomato puree, and the like. Grain plants of asingle species and materials produced from grain plants of a singlespecies, such as corn syrup, rye flour, wheat flour, oat bran, and thelike are also considered to be one component foods. Alternatively, thetwo or more component food preparation can be a mixture of one or moreone component foods and one or more essential nutrients. Preferably, theamount of at least one of the one or more essential nutrients present inthe two component food is greater than the amount of the at least oneessential nutrient that is naturally present collectively in the one ormore one component foods. For example, where the essential nutrients arevitamin X and vitamin Y and where the one component food is orange juiceand where the orange juice naturally contains vitamin X and vitamin Y inamounts “Nx” and “Ny”, respectively, it is preferred that thecomposition contain vitamin X and vitamin Y in amounts “Tx” and “Ty”,respectively, so that Tx is greater than Nx, Ty is greater than Ny, orboth.

[0022] Food preparations particularly well suited to the practice of thepresent invention include breakfast foods, such as prepared cereals,toaster pastries, and breakfast drink mixes; infant formulas; dietarysupplements; complete diet formulas; and weight-loss preparations, suchas weight-loss drinks and weight-loss bars.

[0023] The food preparation can be one which naturally contains nonatural isomer of reduced folate. Alternatively, it can contain anatural molar amount of a natural isomer of reduced folate. For purposesof this application, the molar amount of natural isomer of reducedfolate (i.e., collectively, the number of moles of (6S)-tetrahydrofolicacid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolicacid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof) contained per gram of food is designated “N”. For some foods,the molar amount of natural isomer of reduced folate naturally presentis known. For others, the molar amount of natural isomer of reducedfolate can be determined by a number of sensitive and specific methods,such as microbial growth dependence, folate binding protein basedassays, high-performance liquid chromatography (“HPLC”) and gaschromatography (“GC”) . Suitable methods are described, for example, inCossins, E. A., “Folates in Biological Materials,” in Folates andPterins, Vol. 1, Blakley et al., eds., New York:John Wiley & Sons, pp.1-60 (1984), which is hereby incorporated by reference.

[0024] The molar amount of natural isomer of reduced folate present inthe composition of the present invention is greater that the molaramount of natural isomer of reduced folate present in the foodpreparation. For purposes of this application, the molar amount ofnatural isomer of reduced folate (i.e., collectively, the number ofmoles of (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof) contained per gram of composition is designated “T”. Thus, Tnecessarily must be greater than N. Preferably, T/N is greater than105%; more preferably, it is greater that 110%; and, most preferably, itis greater than 120%.

[0025] As indicated above; the composition can, optionally, include oneor more unnatural isomers of reduced folate. When present in thecomposition, the one or more unnatural isomers of reduced folate ispreferably present in a molar amount which is less than T minus N.

[0026] The present invention also relates to a method for increasing thefolate content of a nutritional substance. The nutritional substance canbe a food preparation, an essential nutrient preparation, or acombination of a food preparation and an essential nutrient preparation.The folate content of the nutritional substance is increased byincorporating one or more natural isomers of reduced folate into or withthe nutritional substance. This can be achieved by methods well known inthe art of food and essential nutrient preparation, such as byhomegenizing, coating, spraying, coarsely mixing, tossing, kneading,pilling, and extruding one or more unnatural isomer of reduced folate,singly or in combination, onto or with the nutritional substance.

[0027] One or more of the one or more natural isomers of reduced folatethat are added to the nutritional substance in accordance with thepresent invention can be substantially chirally pure or each of the oneor more natural isomers of reduced folate can be chirally pure.Alternatively, one or more of the one or more natural isomers of reducedfolate can be present in a mixture with one or more unnatural isomers ofreduced folate. The molar amount of the one or more natural isomers ofreduced folate and the one or more unnatural isomers of reduced folatepresent in the mixture added to the nutritional substance can be thesame, as in the case where a racemic mixture is added, or they can bedifferent. Preferably the molar amount of the natural isomer exceeds themolar amount of the unnatural isomer. Additionally or alternatively,unnatural isomer of reduced folate can be incorporated in a separatestep subsequent to or prior to incorporating the one or more naturalisomers of reduced folate into the nutritional substance. It ispreferred that the collective molar amount of unnatural isomer ofreduced folate added before, during, and/or after the addition ofnatural isomer of reduced folate be less than the collective molaramount of natural isomer added.

[0028] Natural isomers of reduced folates that are substantiallychirally pure can be prepared by any suitable method, including, forexample, by the method described in U.S. Pat. No. 5,350,851 to Bailey etal., which is hereby incorporated by reference.

[0029] When the nutritional substance is a food preparation, in additionto incorporating a natural isomer of reduced folate, one or moreessential nutrients, optionally, can be incorporated into the foodpreparation. The essential nutrients can, for example, be added to thefood preparation before, during, or after addition of the natural isomerof reduced folate.

[0030] The compositions of the present invention can be used to increasea subject's dietary intake of folate by administering the composition tothe subject. The subject can be an animal, such as a dog or a cat;alternatively, the subject can be a human. Certain classes ofindividuals are viewed to be especially benefitted by increasing dietaryintake of folate. These include pregnant females; females who have had amiscarriage; females who have carried a fetus having a neural tubedefect, a cleft lip defect, or a cleft palate defect; and humans whosuffer vascular disease.

[0031] The compositions can also be used to treat a subject afflictedwith intestinal malabsorption, especially folate malabsorption. Whentreating a subject afflicted with intestinal malabsorption, the amountof composition administered is preferably effective to cause an increasein the subject's blood folate level. More preferably, the amountadministered is effective to produce blood folate levels in an normalrange, as determined by conventional blood-folate analysis methods, suchas with the Quanta Phase II assay from BioRad Laboratories, Hercules,Calif.

[0032] The compositions can be administered enterally, such as orally,intragastricly, or transpyloricly. Many factors that may modify theaction of the composition can be taken into account by those skilled inthe art; e.g., body weight, sex, diet, time of administration, route ofadministration, rate of excretion, condition of the subject, andreaction sensitivities and severities. Administration can be carried outcontinuously or periodically, such as once daily, or once with everymeal.

[0033] Compositions containing the natural isomer of reduced folates arepreferably for enteral consumption (enteral including oral,intragastric, or transpyloric), and are of any, food preparation, foodsupplement, essential nutrient preparation, or vitamin preparation.Typical examples of such food or vitamin preparations are those to whichfolic acid (I) is currently added for use by either humans or otheranimals. Natural isomer means a tetrahydrofolate having the naturalconfiguration at both the glutamate a- and the pteridine 6-carbons. Morespecifically, these compositions are, but are not limited to,multivitamin (with or without minerals and other nutrients) preparations(such preparations can be in solid, liquid or suspension forms);breakfast foods such as prepared cereals, breakfast drink mixes, toasterpastries and breakfast bars; infant formulas; dietary supplements andcomplete diet and weight-loss formulas and bars; animal feed or animalfeed supplements (for example, for poultry), and pet foods. The naturalisomer of reduced folates can also be used in products which healthofficials might in the future deem an appropriate vehicles forsatisfaction of the daily folate requirement. The composition of thepresent invention can include a natural isomer of a tetrahydrofolate,such as II-VIII, preferably the monoglutamate form, in a vitaminformulation preferably containing at least one other vitamin (forexample another vitamin such as a B vitamin) other than ascorbic acid(vitamin C), although ascorbic acid can be an additional component. Suchnutrients or vitamins can be those intended for either human or animaluse. Other ingredients may also be present, such as fillers, bindingagents, stabilizers, sweeteners, including nutitive sweeteners (e.g.sucrose, sorbitol and other polyols) and non-nutritive sweeteners (e.g.saccharin, aspartame, and acesulfame K), colorants, flavors, buffers,salts, coatings, and the like that are known to those skilled in the artof vitamin formulation.

[0034] For many applications of the described reduced folates (IIthrough VIII) 5-methyl-(6S)-tetrahydrofolic acid (III),5-formyl-(6S)-tetrahydrofolic acid (IV), and5,10-methenyl-(6R)-tetrahydrofolic acid (VII) are preferred. All of thereduced folates are to differing extent subject to oxidation by air.Several factors influence this susceptibility, foremost beingderivatization of the 5-nitrogen which considerably slows oxidation. The5-methyl- and 5-formyl derivatives are the most abundant forms of folatefound in most tissues of the body. The predominate folate in human bloodis 5-methyl-(6S)-tetrahydrofolic acid (III).

[0035] In using tetrahydrofolates for increasing the folate in anutritional composition, several factors should be considered. Manynutrients are unstable to processing, including several of the vitamins.For example, vitamins A, B₁ (thiamin), and ascorbic acid are especiallylabile under some conditions. Many procedures are known for enhancingthe stability of the various nutrients such as pH and moisture controlof the composition. Components which are to a degree incompatible witheach other, for example ascorbic acid with the more oxidized forms ofiron or copper, can be made to be present heterogeneously in thecomposition. Nutrients which are unstable to heat are often added afterbaking steps; for example, vitamins are often sprayed onto breakfastcereals subsequent to toasting. Nutrients which are unstable to air canbe packaged in a reduced oxygen condition, and/or in containers thathave low or no permeability to oxygen. These and other procedures knownto those skilled in the art are useful for maintaining folates in theirnatural reduced form.

[0036] The rate of oxidation of a reduced folate in the presence of airis increased in water solutions. The shelf life of liquid preparationswill be greatly extended if they are preferably kept in air-tightcontainers. Replacing air with an inert gas such as nitrogen or argonalso retards loss. Reduced folates can also be protected from oxidationby a number of reducing agents and antioxidants, the most relevant ofthese being other vitamins that are often included in multivitaminmixtures or nutritional compositions. Ascorbic acid has been used forprotection of reduced folates in biochemical experiments and proceduresfor the laboratory analysis of biological samples. Such protection neednot be limited to use of ascorbic acid or other vitamins; other agentssuitable for human or animal consumption are useful, for exampleiso-ascorbic acid and certain thiols, such as glutathione. Further,known packaging and formulation technologies which increase thestability of compounds such as ascorbic acid or other air labilematerials (for example, coated forms, blister packaging, and use ofreduced metals or metal complexes) are useful for the maintenance ofreduced folates. The salt form of a reduced folate also somewhat affectsstability and solubility, and this can be optimized for the needs ofeach product. The pH of the final composition can also be optimizedaccording to the stability properties of the particular reduced folatederivative used and of the other components present, as is wellunderstood in the arts of processing nutrients and of folate compounds.For example, in the presence of moisture 5,10-methenyl-(6R)-FH₄ (VII)can be transformed into 10-formyl-(6R)-FH₄ and 5-formyl-(6S)-FH₄ (IV)(the latter also a preferred compound) in a pH dependent manner.Compostions containing 5,10-methenyl-(6R)-FH₄ (VII) are most stable tooxidation when either substantially dry and/or have an acidity less thanabout pH 4. With proper attention to the above factors, the lability ofreduced folates need not limit the life of a product, especially withIII and IV which are more resistant than ascorbic acid to many oxidationreactions.

[0037] The substitution of a reduced folate for folic acid (I) shouldtake into account the differences in molecular weights of the variousforms. For example, the current U.S. Reference Daily Intake of 0.4 mg offolic acid (I) corresponds to 0.91 micromole using an anhydrousmolecular weight of 441.4. The effective molecular weight of reducedfolates depends upon the derivative employed (i.e. II-VIII), the saltform, and water content. For example, 0.91 micromole of5-formyl-(6S)-tetrahydrofolic acid (IV) calcium salt-pentahydrate wouldweigh 0.545 mg, and 0.91 micromole of 5-methyl-(6S)-tetrahydrofolic acid(III) disodium salt would weigh 0.456 mg. Several salt forms of thereduced folates are described in the literature, such as hydrochloride,sodium, potassium, magnesium, calcium, and others and having variouswater content. For each of these forms a similar calculation can bemade. The amount required to achieve the mole equivalent to a desiredfraction of the RDI would then be that fraction of this new weight. Asan example, 25% RDI of 5-methyl-(6S)-tetrahydrofolic acid (III) disodiumsalt would be 0.25×0.456=0.114 mg, the mole equivalent of 0.10 mg offolic acid (I). Previous investigations of groups of individuals havinga normal uptake of folic acid (I) have shown that the bioavailability ofthe reduced folates is similar on a mole basis.

[0038] As mentioned above, loss of nutrients during processing,especially of foods is well known to those skilled in this art. An oftenpracticed procedure is the addition of an initial excess, an “overage”,of a particular nutrient or nutrients, such that the finalpost-processing amount is at the desired level. Many highly sensitiveand specific methods are known (such as microbial growth dependence,folate binding protein based assays, HPLC and GC) for the analysis offolates, in both the reduced and oxidized forms as well as for theirvarious derivatives. These assays permit adjustment of the added amountof the natural isomer of a reduced folate so as to yield the desiredfinal amount subsequent to processing and packaging. The range of thenatural isomer of a reduced folate in the composition of this inventionis preferably that fulfilling between about 5% and about 200% of the RDIof humans for folate, and should be taken to encompass both thesituation where allowance is made for processing loss, and also where nosuch allowance is made. Separate RDA dosages are specified for differentgroups of people, for example pregnant and non-pregnant women. Further,the RDI level although relying on RDA values, can be different from RDAvalues. The above range of “between about 5% and about 200% of the RDIfor folate” should be taken to operate independently on each of theseseparate RDA and RDI specifications, or their foreign equivalents, aspresently stated or as modified in the future. For the purpose of thisinvention these several specifications shall be referred to as the dailyrequirement for folate. Unless the recommended dietary allowance forfolate in humans is increased, the maximum final amount of the naturalisomer of a reduced folate in composition for human use in satisfyingthe daily requirement for folate preferably should not exceed about 4.5micromole per dose or customarily consumed serving. However, forindividuals afflicted with intestinal malabsorption, such as celiacdisease or tropical sprue, compositions containing higher amounts of thenatural isomer of a reduced folate will be useful.

[0039] For the purpose of this invention an essential nutrientcomposition can be a dietary supplement or the like, the substantialfolate component of which is derived from substantially puretetrahydrofolic acid or derivative thereof, such as compounds II throughVIII. Essential nutrient compositions encompassed by this inventioncomprise the natural isomer of a reduced folate preferably within theabove described range along with other vitamins and/or other nutrientswhich are preferably each present in an amount that is considered to besafe. In formulating compositions for animal consumption manufacturersoften considerably exceed the dosage recommended by the NRC for folate(by 10-fold, 20-fold, or more in some cases), not only to overcomelosses during processing, but also to cover occasions of possibleincreased need for folate, such as during antibiotic treatment. Othervitamin and nutrient components can be present in amounts that varyconsiderably from NRC recommendations. The following examples are givento further illustrate the invention, and are not intended to limit itsscope in any way.

EXAMPLES

[0040] 1) A typical ready to eat breakfast cereal: corn (and/or othergrains), sugar, salt, malt flavoring, such that a 30 g serving providesabout 2 g of protein, 26 g total carbohydrate, and 330 mg of sodium,also containing per serving size vitamin A palmitate (15% of RDI),ascorbic acid (25% of RDI), reduced iron (45% of RDI), vitamin D (10% ofRDI), thiamin hydrochloride (25% of RDI), riboflavin (25% of RDI),niacinamide (25% of RDI), pyridoxine hydrochloride (25% of RDI), and0.114 mg of 5-methyl-6(S)-tetrahydrofolic acid (III) disodium salt (themole equivalent of 0.1 mg folic acid, 25% of RDI).

[0041] 2) A typical daily multivitamin tablet: calcium carbonate,ascorbic acid (60 mg, 100% RDI), gelatin, vitamin E acetate (30 I.U.,100% RDI), starch, niacinamide (20 mg, 100% RDI),hydroxypropyl-methylcellulose, calcium pantothenate (10 mg, 100% RDI),calcium silicate, hydroxypropylcellulose, pyridoxine hydrochloride (2mg, 100% RDI), riboflavin (1.7 mg, 100% RDI), thiamin mononitrate (1.5mg, 100% RDI), beta carotene & vitamin A acetate (5000 I.U., 100% RDI),sodium hexametaphosphate, magnesium stearate, vitamin D (400 I.U., 100%RDI), vitamin B₁₂ (6 μg, 100% RDI), lecithin, and 0.437 mg of5-methyl-6(S)-tetrahydrofolic acid (III) magnesium salt (the moleequivalent of 0.4 mg folic acid, 100% of RDI).

[0042] 3) A typical daily multivitamin and minerals tablet: calciumphosphate (130 mg of elemental calcium), magnesium hydroxide & stearate(100 mg, 25% RDI), cellulose, potassium chloride, ascorbic acid (60 mg,100% RDI), gelatin, ferrous fumarate (18 mg elemental iron, 100% RDI),zinc sulfate (15 mg, 100% RDI), modified cellulose gum, vitamin Eacetate (30 I.U., 100% RDI), citric acid, niacinamide (20 mg, 100% RDI),magnesium stearate, hydroxypropyl-methylcellulose, calcium pantothenate(10 mg, 100% RDI), selenium yeast, polyvinylpyrrolidone,hydroxypropylcellulose, manganese sulfate, silica, copper oxide (2 mg,100% RDI), chromium yeast, molybdenum yeast, pyridoxine hydrochloride (2mg, 100% RDI), riboflavin (1.7 mg, 100% RDI), thiamin mononitrate (1.5mg, 100% RDI), beta carotene & vitamin A acetate (5000 I.U., 100% RDI),potassium iodide (150 μg, 100% RDI), sodium hexametaphosphate, biotin(30 μg, 10% RDI), vitamin D (400 I.U., 100% RDI), vitamin B₁₂ (6 μg,100% RDI), lecithin, and 0.545 mg 5-formyl-(6S)-tetrahydrofolic acid(IV) calcium salt-pentahydrate (the mole equivalent of 0.4 mg of folicacid, 100% RDI).

[0043] 4) A typical daily multivitamin and minerals tablet for olderadults: calcium carbonate, calcium phosphate (200 mg Ca, 20% RDI; 48 mgphosphorous, 5% RDI), magnesium oxide, magnesium stearate (100 mg, 25%RDI), potassium chloride (80 mg, 2% RDI), microrystalline cellulose,ascorbic acid (60 mg, 100% RDI), gelatin, d'l-alfa-tocopheryl acetate(45 I.U., 150% RDI), modified food starch, maltodextrin, crospovidone,reduced iron (4 mg, 22 RDI), hydroxypropyl methylcellulose, niacinamide(20 mg, 100% RDI), zinc oxide (15 mg, 100% RDI), calcium pantothenate,manganese sulfate (3.5 mg), vitamin D (400 I.U., 100% RDI), titaniumdioxide, vitamin A and β-carotene (5000 I.U., 100% RDI), stearic acid,pyridoxine hyrochloride (3 mg, 150% RDI), riboflavin (1.7 mg, 100% RDI),silicon dioxide, copper oxide (2 mg, 100% RDI), dextrose, thiaminmononitrate (1.5 mg, 100% RDI), triethyl citrate, polysorbate 80,chhromium chloride (130 μg),, artificial colors, potassium iodide ((150μg, 100% RDI), sodium metasilicate (2 mg), sodium molybdate (160 μg),borates, sodium selenate (20 μg), biotin (30 μg, 10% RDI), sodiummetavanadate (10 μg), cyanocobalamin (25 μg, 417% RDI), nickeloussulfate (5 μg), and phytonadione, and 5,10-methenyl-(6R)-tetrahydrofolicacid hydrochloride (VII)(0.44 mg, the mole equivalent of 0.4 mg of folicacid, 100% RDI).

[0044] 5) A typical complete diet drink: water, sugar, calcium andsodium caseinates, maltodextrin, high-oleic safflower oil, soy protein,soy oil, canola oil, cocoa, sodium and potassium citrates, calciumcarbonate and phosphate (250 mg Ca, 25% RDI), magnesium chloride andphosphate (100 mg Mg, 25% RDI), sodium chloride, soy lecithin, cholinechloride, flavor, ascorbic acid (30 mg, 50% RDI), carrageenan, zincsulfate (5.6 mg, 37% RDI), ferrous sulfate (4.5 mg Fe, 25% RDI),alfa-tocopheryl acetate (11.3 I.U., 37.7% RDI), niacinamide (5 mg, 25%RDI), calcium pantothenate (2.5 mg, 25% RDI), manganese sulfate (1.3mg), copper salt (25% RDI), vitamin A palmitate (1250 I.U., 25% RDI),thiamin hydrochloride (0.375 mg, 25% RDI), pyridoxine hydrochloride (0.5mg, 25% RDI), riboflavin (0.425 mg, 25% RDI), biotin (75 μg, 25% RDI),sodium molybdate (38 μg), chromium chloride (25 μg), potassium iodide(37.5 μg, 25% RDI), sodium selenate (18 μg), phylloquinone (vitamin K₁),cyanocobalamin (1.5 μg, 25% RDI), vitamin D₃ (100 I.U., 25% RDI), and0.136 mg 5-formyl-(6S)-tetrahydrofolic acid (IV) calciumsalt-pentahydrate (the mole equivalent of 0.1 mg of folic acid, 25%RDI), packaged in an air-tight container, and supplying about 225calories.

[0045] 6) A typical enhanced B-vitamin/tetrahydrofolate tablet: dibasiccalcium phosphate, pyridoxine hydrochloride (50 mg, 2,500% RDI),cellulose, stearic acid, magnesium stearate, and 0.912 mg of5-methyl-6(S)-tetrahydrofolic acid (III) disodium salt (the moleequivalent of 0.8 mg folic acid, 200% of RDI for adults, 100% RDA forpregnant women).

[0046] 7) A typical poultry feed vitamin supplement: (amounts per kg ofdiet) vitamin A (trans retinyl acetate, 5500 I.U.), vitamin E (11 I.U.),menadione sodium bisulfite (1.1 mg), vitamin D₃ (1100 I.U.), riboflavin(4.4 mg), vitamin B₁₂ (10 μg), vitamin B₆ (3.0 mg), thiamin mononitrate(2.2 mg), biotin (0.3 mg), ethoxyquin (125 mg), and 2.0 mg5-formyl-(6S)-tetrahydrofolic acid (IV) calcium salt-pentahydrate (themole equivalent of 1.45 mg of folic acid).

[0047] 8) A typical dry cat food: ground yellow corn, corn gluten meal,soybean meal, poultry by-product meal, animal fat, fish meal, meat andbone meal, ground wheat, phosphoric acid calcium carbonate, dried animaldigest, salt, brewers dried yeast, potassium chloride, dried wheysolubles, choline chloride, dried skimmed milk, taurine, L-lysine, zincoxide, ferrous sulfate, niacin, vitamin A, vitamin D₃, vitamin B₁₂,calcium pantothenate, citric acid, manganese sulfate, riboflavinsupplement, biotin, copper salt, thiamine mononitrate, pyridoxinehydrochloride, menadione sodium bisulfate complex, such that the crudeprotein is not less than 31%, crude fat is not less than 8%, crude fiberis not more than 4.5%, moisture is not more than 12%, calcium is notless than 1.2%, phosphorous is not less than 1.0%, sodium chloride isnot more than 1.5%, the metabolizable energy is about 3,600 kcal/kg,taurine, iron, vitamins A, D₃, B₁₂, and E are at least 100% of levelsrecommended by the Association of American Feed Control Officials, andcontaining not less than 0.97 mg/kg diet 5-methyl-6(S)-tetrahydrofolicacid (III) calcium salt dihydrate (the mole equivalent of 0.8 mg/kg dietof folic acid).

[0048] 9) A typical soy based infant formula: 75.5% water; 13% sucrose;6.6% oleo oil: coconut, high oleic (safflower or sunflower), and soybeanoils; 3.8% soy protein isolate; (protein 2.7 g, fat 5.3 g, carbohydrate10.2, linoleic acid 500 mg); potassium citrate and bicarbonate(potassium 105 mg); monobasic potassium and dibasic calcium phosphates(phosphorous 63 mg); soy lecithin; taurine; calcium carrageenan; calciumhydroxide, chloride and citrate (calcium 90 mg); sodium chloride (sodium30 mg); L-methionine; zinc (Zn 0.8 mg), ferrous (Fe 1.8 mg), andmanganese (Mn 30 μg) sulfates; copper salt (Cu 70 μg); taurine;L-carnitine; potassium iodide (I 9 μg); ascorbic acid (8.3 mg); cholinechloride; alpha-tocopheryl acetate (1.4 I.U.); niacinamide (750 μg);vitamin A palmitate and betacarotene (300 I.U.); calcium pantothenate(450 μg); thiamin hydrochloride (100 μg); riboflavin (150 μg);pyridoxine hydrochloride (62.5 μg); vitamin K, (15 μg); biotiri (5.5μg); vitamin D₃ (60 I.U.); cyanocobalamin (0.3 μg); and 9.1 μg of5-methyl-6(S)-tetrahydrofolic acid (III) calcium salt dihydrate (themole equivalent of 7.5 μg of folic acid), packaged in an air-tightcontainer (amounts are per 150 ml of 1:1 diluted formula).

[0049] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned. While the above description contains manyspecificities, these should not be construed as limitations on the scopeof the invention, but rather as an exemplification of preferredembodiments thereof. Changes therein and other uses will occur to thoseskilled in the art which are encompassed within the spirit of theinvention as defined by the scope of the claims and their legalequivalents.

What is claimed:
 1. A composition comprising: one or more naturalisomers of reduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; a nutritional substance selected from the group consisting of afood preparation, an essential nutrient preparation, and combinationsthereof; wherein, when the nutritional substance is a food preparation,the food preparation comprises two or more food components and each gramof said food preparation has a natural molar amount, N, of said one ormore natural isomers of reduced folate, wherein N is greater or equal tozero and wherein each gram of said composition has a total molar amount,T, of said one or more natural isomers of reduced folate greater than N;and wherein, when the nutritional substance is an essential nutrientpreparation, the essential nutrient preparation comprises a vitaminother than ascorbic acid.
 2. A composition according to claim 1 ,wherein the one or more natural isomers of reduced folate is selectedfrom the group consisting of 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolicacid, and polyglutamyl derivatives thereof.
 3. A composition accordingto claim 1 , wherein the total molar amount of said one or more naturalisomers of reduced folate is between 5% and 200% of a human dailyrequirement for folate per customarily consumed quantity of saidcomposition.
 4. A composition according to claim 1 , wherein the totalmolar amount of said one or more natural isomers of reduced folate isbetween 5% and 3000% of an animal daily requirement for folate percustomarily consumed quantity of said composition.
 5. A compositionaccording to claim 1 , wherein said nutritional substance is a foodpreparation.
 6. The composition according to claim 5 , wherein thenutritional substance is a food preparation and wherein each gram ofsaid food preparation further comprises no unnatural isomers of reducedfolate selected from the group consisting of (6R)-tetrahydrofolic acid,5-methyl-(6R)-tetrahydrofolic acid, 5-formyl-(6R)-tetrahydrofolic acid,10-formyl-(6S)-tetrahydrofolic acid, 5,10-methylene-(6S)-tetrahydrofolicacid, 5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, or one or more of said unnatural isomers of reduced folate in amolar amount less than T minus N.
 7. A composition according to claim 5, wherein the food preparation is selected from the group consisting ofbreakfast foods, infant formulas, dietary supplements, complete dietformulas, and weight-loss preparations.
 8. A composition according toclaim 7 , wherein the breakfast food is a prepared cereal, a breakfastdrink mix, or a toaster pastry, and wherein the weight-loss preparationsis a weight-loss drink or a weight-loss bar.
 9. A composition accordingto claim 1 , wherein the nutritional substance is an essential nutrientpreparation comprising a vitamin other than ascorbic acid.
 10. Acomposition according to claim 9 , wherein the essential nutrientpreparation further comprises ascorbic acid.
 11. A composition accordingto claim 9 , wherein the vitamin is present in an amount equal to orgreater than 25% of the daily requirement for the vitamin percustomarily consumed quantity of said essential nutrient preparation.12. A method for increasing the folate content of a nutritionalsubstance comprising: providing a nutritional substance selected fromthe group consisting of a food preparation, an essential nutrientpreparation, and combinations thereof; and incorporating into thenutritional substance a molar amount of one or more natural isomers ofreduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; wherein, when the nutritional substance is a -food preparation,the food preparation comprises two or more food components; and wherein,when the nutritional substance is an essential nutrient preparation, theessential nutrient preparation comprises a vitamin other than ascorbicacid.
 13. A method according to claim 12 further comprisingincorporating into the nutritional substance a molar amount of one ormore unnatural isomers of reduced folate selected from the groupconsisting of (6R)-tetrahydrofolic acid, 5-methyl-(6R)-tetrahydrofolicacid, 5-formyl-(6R)-tetrahydrofolic acid, 10-formyl-(6S)-tetrahydrofolicacid, 5,10-methylene-(6S)-tetrahydrofolic acid,5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, wherein the molar amount of the one or more unnatural isomersof reduced folate is less than the molar amount of the one or morenatural isomers of reduced folate.
 14. A method according to claim 12 ,wherein each of the one or more natural isomers of reduced folate issubstantially chirally pure.
 15. A method according to claim 12 ,wherein the one or more natural isomers of reduced folate is selectedfrom the group consisting of 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolicacid, and polyglutamyl derivatives thereof.
 16. A method according toclaim 12 , wherein the nutritional substance is an essential nutrientpreparation comprising a vitamin other than ascorbic acid.
 17. A methodaccording to claim 16 , wherein the essential nutient preparationfurther comprises ascorbic acid.
 18. A method according to claim 12 ,wherein the nutritional substance is a food preparation and wherein saidmethod further comprises: incorporating a vitamin into the foodpreparation.
 19. A method for increasing a subject's dietary intake offolate comprising: administering a composition according to claim 1 tothe subject.
 20. A method according to claim 19 , wherein saidadministering is carried out by enteral administration.
 21. A methodaccording to claim 19 , wherein the subject is an animal.
 22. A methodaccording to claim 21 , wherein the total molar amount of said one ormore natural isomers of reduced folate is between 5% and 3000% of theanimal's daily requirement for folate per customarily consumed quantityof said composition.
 23. A method according to claim 19 , wherein thesubject is a human.
 24. A method according to claim 23 , wherein thetotal molar amount of said one or more natural isomers of reduced folateis between 5% and 200% of the human's daily requirement for folate percustomarily consumed quantity of said composition.
 25. A methodaccording to claim 23 , wherein the human is selected from the groupconsisting of a pregnant female; a female who has had a miscarriage; afemale who has carried a fetus having a neural tube defect, a cleft lipdefect, or a cleft palate defect; and a human who suffers vasculardisease.
 26. A method for treating a subject afflicted with intestinalmalabsorption comprising: administering to the subject an amount of acomposition according to claim 1 effective to increase the subject'sblood folate level.